Glucansucrases, commonly known as glucosyltransferases, found in lactic acid bacteria (Streptococci, Leuconostoc sp., Lactococcus sp., and Lactobacillus sp.), are enzymes belonging to the glycosidase and transglycosidase of glycoside-hydrolase family 70 that catalyze the transfer of glucosyl units from the cleavage of sucrose to a growing α-glucan chain (Henrissat, B. Biochem. Soc. Trans. 26, 153-156 (1998)). The nature of the linkages between glucosyl units determines the water solubility and properties of the glucan. Thus, a higher content of an α-1,3 linkage results in greater insolubility (Monchois et al FEMS Microbiol. Rev. 23, 131-151 (1999)). In the presence of acceptor molecules, such as maltose, glucansucrases can catalyze the synthesis of low molecular weight oligosaccharides.
Glucansucrases have industrial value because of the production of glucans and oligosaccharides of biologically importance. They play a key role in cariogenic processes and thus can be used in the development of vaccines against caries. More specifically, glucansucrases synthesize glucans, which are of central importance in adhesive interactions in plaque, where they mediate attachment of bacteria to the tooth surface and to other bacteria, thus stabilizing the plaque biofilm, serve as energy stores aiding the survival of plaque bacteria and modulating the permeability of plaque and hence the acid level at the enamel surface (Colby et al Soc. J. Appl. Microbiol. Symp. Suppl., 83, 80S-88S (1991)).
Alternansucrase is a large glucansucrase having 2,057 amino acids that, in the absence of external acceptors and starting from sucrose, catalyzes the formation of fructose and an unusual polymer consisting of glucopyranosyl residues alternatively linked by α-1,6 and α1,3 osidic bonds, called alternan. The polysacharide alternan was first described by Jeanes et al J. Am. Chem Soc, 76, 5041-5052 (1954) as one of two extracellular α-D glucans, referred to as fraction S, produced by Leuconostoc mesenteroides NRRL B-1355. Since the α-1,3-linkages are part of the linear chain of the S fraction and there are not any conservative α-1,6 linkages, this fraction was not considered a true dextran, but was named alternan by Côté and Robyt (Carbohydrate Res. 101, 57-74 (1982)).
In the presence of external acceptors, such as for instance, maltose, isomaltose, isomaltriose and methyl-α-D-glucan and cellobiose, alternansucrase catalyzes at the acceptors the synthesis of α-D-glucan chains, in which the glucose moieties are predominantly alternating linked by α-1,6 and α1,3 glycosidic bonds and release of fructose. Depending on the acceptor used, the resulting products have different structures and molecular weights that are lower than high molecular weight alternan. They have a polymerization degree of less than 15. Because of the polymerization degree, these products are often referred to as oligoalternans (Pelenc et al, Sciences Des Aliments 11, 465-476 (1991)). In the preparation of oligoalternans using alternansucrase, maltose is an acceptor producing high oligoalternan yields, while panose is the first acceptor product which is formed starting from maltose through the formation of α-1,6 glycosidic bonds (Lopez-Mungia et al Enzyme Microb. Technol. 15, 77-85 (1993)).
Because of its physico-chemical properties (high solubility and low viscosity) alternan has valuable use in the pharmaceutical industry, for instance, as a carrier of pharmaceutically active ingredients or as blood plasma extenders. Also alternans have been suggested as additives in the textile, cosmetics and food industry and in particular as prebiotics. (Lopez-Munguia et al Enzyme Microb, Technol. 15 (1993). Besides acting as an additive, alternan can be used as a substitute for gum Arabic (Côté, Carbohydrate Polymers 19, 249-252 (1992)).
Alternan is generally prepared in a cell-free system using partially purified proteins or by fermentation using alternansucrase-producing strains of Leuconostic mesenteroides. Various purification methods for alternansucrases have been previously described (Lopez-Mungia et al, Enzyme Microb, Technol. 15, 77-85 (1993) Côté and Robyt, Carbohydrate Research 101, 57-74 (1982)). These methods are however complex, relatively costly and lead to very low protein yields.
Moreover, since the alternansucrase produced in the fermentation methods is not highly pure, dextran impurities are generally present in the alternan produced. Moreover, the enzyme production is induced by sucrose and the protein extracts are contaminated by the co-synthesized enzymes. To separate the dextran and other impurities from the alternan is relatively time-consuming and costly.
Alternative methods have been suggested such as the production of alternansucrase by recombinant means. In fact the alternansucrase gene was in fact cloned in E. coli, but the level of expression was extremely low, 160 U.1−1 compared to the native 1,730 U.1−1. Moreover, no information about the quality of the expressed product was reported (Arguello-Morales et al; FEMS Microbiol. Lett. 182, 81-85 (2000)). Furthermore, the expressed enzyme was highly degraded due to its expression in E. coli. 
U.S. Pat. No. 6,570,065 describes methods for preparing transgenic plants which synthesize alternan due to the insertion of nucleic acid molecules encoding an alternansucrase. Also described in this patent application is the production of alternansucrase in E. coli. However, the full length DNA sequence coding for alternansucrase was used and hence the yields produced were low.
In view of the above, there is a need in this art to produce a highly purified and enzymatically active alternansucrase, which can be used to produce alternans and oligoalternans.
Thus, it is an object of the present invention to overcome the problems associated with the prior art.
It is another object of the present invention to provide a recombinantly produced alternansucrase that retains its enzyme activity, which has practically neither dextran nor dextransucrase impurities.
Another object of the present invention is to provide nucleic acid sequences of truncated and mutated alternansucrases, vectors and host cells transformed by the vectors.
Yet another object of the present invention is to provide amino acid sequences of truncated and mutated alternansucrases.
In another object, the present invention provides truncated variants of alternansucrase, which are better expressed and less degraded compared to the full length alternansucrase.
In yet another object, the present invention provides mutated alternansucrases which, when subject to an external acceptor synthesizes a large quantity of specific oligosaccharides such as oligodextrans and oligoalternans.
In still another object, the present invention provides a process for producing highly purified alternansucrases, which retain their catalytic activity.
In still another object, the present invention provides a composition comprising truncated or mutated alternansucrases and a pharmaceutically acceptable vehicle.
These and other objects are achieved by the present invention as evidenced by the summary of the invention, description of the preferred embodiments and the claims.